Oxidized glutathione fermentation using Saccharomyces cerevisiae engineered for glutathione metabolism

Abstract

Glutathione is a valuable tripeptide that is widely used in the pharmaceutical, food, and cosmetic industries. Intracellular glutathione exists in two forms, reduced glutathione (GSH) and oxidized glutathione (GSSG). Most of the glutathione produced by fermentation using yeast is in the GSH form because intracellular GSH concentration is higher than GSSG concentration. However, the stability of GSSG is higher than GSH, which makes GSSG more advantageous for industrial production and storage after extraction. In this study, an oxidized glutathione fermentation method using Saccharomyces cerevisiae was developed by following three metabolic engineering steps. First, over-expression of the glutathione peroxidase 3 (GPX3) gene increased the GSSG content better than over-expression of other identified peroxidase (GPX1 or GPX2) genes. Second, the increase in GSSG brought about by GPX3 over-expression was enhanced by the over-expression of the GSH1/GSH2 genes because of an increase in the total glutathione (GSH + GSSG) content. Finally, after deleting the glutathione reductase (GLR1) gene, the resulting GPX3/GSH1/GSH2 over-expressing ΔGLR1 strain yielded 7.3-fold more GSSG compared with the parental strain without a decrease in cell growth. Furthermore, use of this strain also resulted in an enhancement of up to 1.6-fold of the total glutathione content compared with the GSH1/GSH2 over-expressing strain. These results indicate that the increase in the oxidized glutathione content helps to improve the stability and total productivity of glutathione.

Improvement of oxidized glutathione fermentation by thiol redox metabolism engineering in Saccharomyces cerevisiae

“…Previously, we reported that an S. cerevisiae strain over-expressing GSH1/GSH2 constructed by a cocktail δ-integration method (Yamada et al 2010) showed higher total glutathione content compared with the parental strain (Hara et al 2012). Recently, we also showed that the deletion of the GLR1 gene was critical to enhancing the GSSG content (Kiriyama et al 2013). Therefore, in this study, we used the GCIΔGLR1 strain as platform strain for further improvement of GSSG production.…”

“…In S. cerevisiae, Glr1 has been identified as a glutathione reductase (Grant and Dawes 1996). Recently, we constructed a GCIΔGLR1 strain by deleting the GLR1 gene from the genome of the GCI strain, which increased the GSSG/GSH ratio by about 2.2-fold compared with the GCI strain (Kiriyama et al 2013). Therefore, the combined effect of over-expression of the thiol oxidase and deletion of glutathione reductase would be expected to synergistically increase the GSSG content of S. cerevisiae.…”

“…The integrated numbers of GSH1 genes and GSH2 genes in the GCI strain were 7 and 14, respectively (Hara et al 2012). A GLR1 gene encoding glutathione reductase Glr1 was deleted from the S. cerevisiae genomic DNA of the GCI strain by homologous replacement (Kiriyama et al 2013). pGK406-ERV1, pGK406-ERV2, and pGK406-ERO1 were digested with NcoI or EcoRV and transformed into the GCIΔGLR1 strain to construct the GCIΔGLR1/ERV1, GCIΔGLR1/ERV2, and GCIΔGLR1/ ERO1 strains, respectively.…”

“…However, the chemical oxidization requires a large amount of acid and it is problem for increasing production cost and environment. We previously reported that GSSG content was increased in strains lacking glutathione reductase and over-expressing Gsh1 and Gsh2 (Kiriyama et al 2013).…”

ATP synthesis in the energy metabolism pathway: a new perspective for manipulating CdSe quantum dots biosynthesized in <em>Saccharomyces cerevisiae</em>

“…36 It was reasonable to hypothesize that the ATP effects may be due to its influence on the synthesis of GSH with its important role in our system. 26,27 Interestingly, when the GSH contents were determined in the Δatp1 strain, the tendency of GSH levels increasing with fluorescence intensities, referred to above, did not appear ( Figure 3A).…”